A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Abstract

Ca 2؉ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca 2؉ pulse. The G293S and G283S mutations delayed the dissipation of Ca 2؉ transients induced in CHO cells by InsP3. In organotypic cultures, Ca 2؉ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca 2؉ transients induced by Ca 2؉ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.hereditary hearing loss ͉ mutations ͉ calcium homeostasis ͉ calcium transport T he receptive organelle of sensory hair cells in the mammalian cochlea, the stereocilia bundle, protrudes from the cell's apical surface. Inner hair cells transduce mechanical vibrations into electrical signals that are eventually transmitted to the brain to be transformed into hearing signals (1), whereas outer hair cells (OHCs) (2) amplify the vibrations of the basilar membrane (3). Mechanical stimuli that are detected as excitatory deflect a hair bundle, thus increasing tension in the tip link, a filament stretched between the tops of stereocilia. This tension is conveyed to mechanosensitive transduction (MET) channels that open to allow ions into the cell (4). The apical surface of hair cells is bathed in endolymph, which is rich in K ϩ but low in Na ϩ and Ca 2ϩ (5). K ϩ carries most the transduction current, but MET channels are Ca 2ϩ selective, i.e., Ca 2ϩ influx is significant even at the low Ca 2ϩ levels of the endolymph, which are much lower than those of other extracellular fluids (6-10): 20-23 M in the rodent cochlea (11, 12), 200-250 M in the vestibular system, possibly because of the presence there of calcium carbonate crystals (13,14). Approximately 10% of the MET current may actually be carried by Ca 2ϩ ions (15). Ca 2ϩ entering through MET channels is rapidly sequestered by buffers in the stereocilia (16,17) and is shuttled back to endolymph by the plasma membrane Ca 2ϩ pump (PMCA) (18,19), which is very concentrated in the stereocilia membrane (Ϸ2,000 per squared micrometer) (15, 19-21). The PMCA is assumed to increase Ca 2ϩ in the immediate pro...

Functional analysis of pancreatitis-associated missense mutations in the pancreatic secretory trypsin inhibitor (SPINK1) gene

“…This issue has been addressed in our previous study. 22 Here, we mention three points: (i) the CHO cell line is one of the most widely used model systems to characterize human secretory proteins (eg see two recent publications 31,32 ), (ii) Western blotting analysis of the three missense mutations (ie p.L12F, p.L14P and p.L14R) that have occurred within the signal peptide of PSTI in the CHO line, yielded similar results to those obtained independently in the human embryonic kidney 293T cell line 22 and (iii) that the N34S polymorphism did not cause any significant reduction of PSTI expression in the CHO cells concurred with the analysis using Saccharomyces cerevisiae BJ1991 as a model system. 20 What is the mechanism underlying the significantly reduced or abolished expression of PSTI in the p.G48E, p.D50E, p.Y54H, p.R65Q, and p.R67C mutants?…”

A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations

“…The proband also carried two missense mutations in the LAMA1 gene coding for laminin subunit 1␣. This is an interesting finding as it recalls the digenic mechanism described in the case of hereditary human deafness, where the mutations of the PMCA2 pump were accompanied by mutations of cadherin-23, a protein involved in the mechanoelectrical transduction process (7,9).…”

“…The hereditary deafness caused by mutations of the PMCA2 isoform (7,9) can be associated with ataxic symptoms (10,11). An ataxia-causing defect was recently described in PMCA3 (12).…”

“…Genetic defects of the PMCA pumps have been described for PMCA2 and PMCA3. A series of mutations of the former in mice and humans cause deafness (7)(8)(9). Interestingly, the deafness phenotype was not the only associated phenotype: one of the PMCA2-defective mice also had prominent ataxic symptoms (10).…”

Alternative splice variants of plasma membrane calcium-ATPases in human corneal epithelium

“…Targeted ablation of PMCA1 in mice leads to embryonic lethality, whereas PMCA4 knockout in mice leads to infertile males (Prasad et al 2007). PMCA2 mutations or knockout cause deafness and ataxia in several animals including humans (Ficarella et al 2007;Kozel et al 1998;Street et al 1998). To date, PMCA deficits have not been documented in any corneal pathology.…”